Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand.
Molecular basis of rare aminoglycoside susceptibility and pathogenesis of Burkholderia pseudomallei clinical isolates from Thailand.
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DOI：
10.1371/journal.pntd.0000519
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发表时间：
2009-09-22
影响因子：
3.8
通讯作者：
Schweizer HP
中科院分区：
文献类型：
--
作者：
Trunck LA;Propst KL;Wuthiekanun V;Tuanyok A;Beckstrom-Sternberg SM;Beckstrom-Sternberg JS;Peacock SJ;Keim P;Dow SW;Schweizer HP
类鼻疽伯克霍尔德菌对氨基糖苷类和大环内酯类具有内在耐药性，主要是由于AmrAB-OprA外排泵表达。我们研究了泰国菌株708 a、2188 a和3799 a对氨基糖苷类药物敏感的分子机制。qRT-PCR显示708 a中不存在amrB转录物，2188 a和3799 a中的水平大大降低。连续传代增加庆大霉素浓度产生2188 a和3799 a突变体，成为同时耐其他氨基糖苷类和大环内酯类，而这样的突变体不能获得与708 a。转录本分析显示，2188 a和3799 a突变体的抗性是由于amrAB-oprA表达通过未知机制上调所致。PCR步移策略的使用揭示了amrAB-oprA操纵子在708 a中缺失，并且这种缺失与超过70 kb的遗传物质的缺失有关。从708 a fosmid文库中拯救amrAB-oprB区域并测序显示存在大的1号染色体缺失（与菌株K96243和1710 b相比分别为131 kb和141 kb）。这种缺失不仅去除了amrAB-oprA操纵子，还去除了用于合成malleobactin和钴胺素的整个基因簇。其他基因删除包括厌氧精氨酸脱亚胺酶途径，推定的1型菌毛和分泌几丁质酶。全基因组测序和PCR分析证实这些基因从708 a中缺失。尽管缺少几个推定的毒力基因，708 a是完全有毒的小鼠类鼻疽模型。菌株708 a可以是遗传操作实验的天然候选物，所述遗传操作实验使用选择剂顺应性抗生素进行选择，并验证实验室构建的Δ（amrAB-oprA）突变体在此类实验中的使用。 类鼻疽伯克霍尔德氏菌是一种新兴的热带疾病类鼻疽的病原体。由于B. B具有感染剂量低、宿主范围广、固有的抗生素耐药性和作为生物武器的历史先例等特点，因此，B.在美国，类鼻疽被美国疾病控制和预防中心以及国家过敏和传染病研究所列为生物防御关注的选择剂和优先病原体。该细菌的抗生素耐药性和/或敏感性和毒力的机制还不清楚。大多数临床和环境B。鼻疽假单胞菌分离株对氨基糖苷类高度耐药，但确实存在敏感变异体。我们对来自泰国的三种这样的变体的研究结果表明，缺乏表达或缺失外排泵是导致这种易感性的原因。一个菌株中存在的大缺失不仅去除了外排泵，还去除了几个推定的毒力基因，包括整个铁载体基因簇。尽管存在这种缺失，但该菌株在急性小鼠类鼻疽模型中具有完全毒性。总之，我们的研究结果阐明了抗生素耐药性和发病机制。他们还验证了先前提倡的在遗传操作实验中使用实验室构建的氨基糖苷类敏感外排泵突变体。
Burkholderia pseudomallei is intrinsically resistant to aminoglycosides and macrolides, mostly due to AmrAB-OprA efflux pump expression. We investigated the molecular mechanisms of aminoglycoside susceptibility exhibited by Thai strains 708a, 2188a, and 3799a. qRT-PCR revealed absence of amrB transcripts in 708a and greatly reduced levels in 2188a and 3799a. Serial passage on increasing gentamicin concentrations yielded 2188a and 3799a mutants that became simultaneously resistant to other aminoglycosides and macrolides, whereas such mutants could not be obtained with 708a. Transcript analysis showed that the resistance of the 2188a and 3799a mutants was due to upregulation of amrAB-oprA expression by unknown mechanism(s). Use of a PCR walking strategy revealed that the amrAB-oprA operon was missing in 708a and that this loss was associated with deletion of more than 70 kb of genetic material. Rescue of the amrAB-oprB region from a 708a fosmid library and sequencing showed the presence of a large chromosome 1 deletion (131 kb and 141 kb compared to strains K96243 and 1710b, respectively). This deletion not only removed the amrAB-oprA operon, but also the entire gene clusters for malleobactin and cobalamin synthesis. Other genes deleted included the anaerobic arginine deiminase pathway, putative type 1 fimbriae and secreted chitinase. Whole genome sequencing and PCR analysis confirmed absence of these genes from 708a. Despite missing several putative virulence genes, 708a was fully virulent in a murine melioidosis model. Strain 708a may be a natural candidate for genetic manipulation experiments that use Select Agent compliant antibiotics for selection and validates the use of laboratory-constructed Δ(amrAB-oprA) mutants in such experiments. Burkholderia pseudomallei is the etiologic agent of melioidosis, an emerging tropical disease. Because of low infectious dose, broad-host-range infectivity, intrinsic antibiotic resistance and historic precedent as a bioweapon, B. pseudomallei was listed in the United States as a Select Agent and Priority Pathogen of biodefense concern by the US Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases. The mechanisms governing antibiotic resistance and/or susceptibility and virulence in this bacterium are not well understood. Most clinical and environmental B. pseudomallei isolates are highly resistant to aminoglycosides, but susceptible variants do exist. The results of our studies with three such variants from Thailand reveal that lack of expression or deletion of an efflux pump is responsible for this susceptibility. The large deletion present in one strain not only removes an efflux pump but also several putative virulence genes, including an entire siderophore gene cluster. Despite this deletion, the strain is fully virulent in an acute mouse melioidosis model. In summary, our findings shed light on mechanisms of antibiotic resistance and pathogenesis. They also validate the previously advocated use of laboratory-constructed, aminoglycoside susceptible efflux pump mutants in genetic manipulation experiments.
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影响因子：
48
作者：
Craig, David W.;Pearson, John V.;Szelinger, Szabolcs;Sekar, Aswin;Redman, Margot;Corneveaux, Jason J.;Pawlowski, Traci L.;Laub, Trisha;Nunn, Gary;Stephan, Dietrich A.;Homer, Nils;Huentelman, Matthew J.
通讯作者：
Huentelman, Matthew J.
影响因子：
3.2
作者：
DeShazer, D;Brett, PJ;Woods, DE
通讯作者：
Woods, DE
影响因子：
4.4
作者：
Barrett, Ashley R.;Kang, Yun;Hoang, Tung T.
通讯作者：
Hoang, Tung T.
影响因子：
3
作者：
Brook, I;Frazier, EH
通讯作者：
Frazier, EH
影响因子：
3.2
作者：
Alice, AF;López, CS;Crosa, JH
通讯作者：
Crosa, JH